Polydisperse native pseudomonas flagellar (H) antigens (FAg) and methods of producing them

Abstract

Polydisperse native Pseudomonas flagellar (H) antigens (FAg) and methods of producing them are described, wherein each monomeric component contains certain amino acids, has a certain N-terminal amino acid sequence and a certain molecular weight and is free from pyrogenic substances. The ratio of the individual amino acids in the flagellar antigen of the individual H-serotypes is stated. For producing the polydisperse native Pseudomonas flagellar (H) antigens (FAg) methods are indicated, wherein Pseudomonas aeruginosa bacterial cultures or fractions thereof are treated with a detergent and the flagellar antigens are separated from the cultures. Prior to treatment or in the presence of the detergent, the bacterial culture is subjected to a shearing procedure, i.e. exposed to shearing forces. Thereafter, the flagellar antigens separated from the bacterial mass are isolated by a chromatographic treatment. For this chromatographic treatment a molecular sieve can be used and a further purification by chromatography on a column can be carried out for obtaining flagellar antigens of high purity.

We claim: 
     
       1. Polydisperse native Pseudomonas flagellar (H) antigen in polymeric form having (i) a diffusion coefficient of D =8.5×10 -9  (cm 2  /sec) (ii) a hydrodynamic radius of R=2.5×10 -5  cm, (iii) a density of d=1.28 g/cm 3 , and (iv) substantial freedom from LPS, said antigen being comprised of monomeric components, each monomeric component (a) containing the following amino acids: aspartic acid Asp), threonine (Thr), serine (Ser), glutamic acid Glu), glycine (Gly), alanine (Ala), valine (Val), soleucine (Ile), leucine (Leu) tyrosine (Tyr), phenyl lanine (Phe), lysine (Lys), and arginine (Arg)   (b) having the N-terminal amino acid sequence alanine (Ala) leucine (Ldu) - threonine (Thr) - valine (Val) - asparagine Asn) - threonine (Thr) - asparagine (Asn) - isoleucine Ile) - alanine (Ala),   (c) having a molecular weight of from 43,500 to 53,050 and   (d) being free from proline, methionine, semicystine and istidine.   
     
     
       2. A flagellar (H) antigen of the H-serotype a 0 , a 3 , a 4  according to claim 1 wherein the monomeric form contains the amino acids: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenyl alanine, lysine and arginine at a ratio of 64:33:35:42:44:68:29:29:37:3:10:19:15 and has a molecular weight of 43,500. 
     
     
       3. A flagellar (H) antigen of the H-serotype a 0 , a 3  according to claim 1 wherein the monomeric form contains the amino acids: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenyl alanine, lysine and arginine at a ratio of 60:35:38:44:47:73:30:30:60:3:12:21:16 and has a molecular weight of 46,700. 
     
     
       4. A flagellar (H) antigen of the H-serotype a 0  according to claim 1 wherein the monomeric form contains the amino acids: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenyl alanine, lysine and arginine at a ratio of 74:50:49:49:49:89:37:29:44:5:14:17:16 and has molecular weight of 52,720. 
     
     
       5. A flagellar (H) antigen of the H-serotype b according to claim 1 wherein the monomeric form contains the amino acids: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenyl alanine, lysine and arginine at a ratio of 74:48:48:49:51:91:38:30:43:4:13:18:18 and has a molecular weight of 53,050. 
     
     
       6. A flagellar (H) antigen of the H-serotype a 0 , a 1 ,a 2  according to claim 1 wherein the monomeric form contains the amino acids: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenyl alanine, lysine and arginine at a ratio of 76:44:40:52:50:81:32:32:41:4:12:20:18 and has a molecular weight of 51,250. 
     
     
       7. A flagellar (H) antigen of the H-serotype a 0 , a 2  according to claim 1 wherein the monomeric form contains the amino acids: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenyl alanine, lysine and arginine at a ratio of 68:41:37:46:44:73:29:29:37:3:10:16:16 and has a molecular weight of 45,900. 
     
     
       8. The flagellar (H) antigen according to claim 1 wherein each monomeric component contains tryptophane (Trp).